Diane Niño

Early Patterns of Gene Expression Correlate With the Humoral Immune Response to Influenza Vaccination in Humans

BACKGROUND Annual vaccination is the primary means for preventing influenza. However, great interindividual variability exists in vaccine responses, the cellular events that take place in vivo after vaccination are poorly understood, and appropriate biomarkers for vaccine responsiveness have not been developed. METHODS We immunized a cohort of healthy male adults with a licensed trivalent influenza vaccine and performed a timed assessment of global gene expression before and after vaccination. We analyzed the relationship between gene expression patterns and the humoral immune response to vaccination. RESULTS Marked up regulation of expression of genes involved in interferon signaling, positive IL-6 regulation, and antigen processing and presentation, were detected within 24 hours of immunization. The late vaccine response showed a transcriptional pattern suggestive of increased protein biosynthesis and cellular proliferation. Integrative analyses revealed a 494-gene expression signature--including STAT1, CD74, and E2F2--which strongly correlates with the magnitude of the antibody response. High vaccine responder status correlates with increased early expression of interferon signaling and antigen processing and presentation genes. CONCLUSIONS The results highlight the role of a systems biology approach in understanding the molecular events that take place in vivo after influenza vaccination and in the development of better predictors of vaccine responsiveness.

A randomized clinical trial of an inactivated avian influenza A (H7N7) vaccine.

Background Concern for a pandemic caused by a newly emerged avian influenza A virus has led to clinical trials with candidate vaccines as preparation for such an event. Most trials have involved vaccines for influenza A (H5N1), A (H7N7) or A (H9N2). Objective To evaluate dosage-related safety and immunogenicity of an inactivated influenza A (H7N7) vaccine in humans. Design One hundred twenty-five healthy young adults were randomized to receive two doses intramuscularly of placebo or 7.5, 15, 45 or 90 µg of HA of an inactivated subunit influenza A (H7N7) vaccine (25 per group), four weeks apart. Reactogenicity was evaluated closely for one week and for any adverse effect for six months after each dose. Serum hemagglutination-inhibiting and neutralizing antibody responses were determined four weeks after each dose and at six months. Results Reactogenicity evaluations indicated the vaccinations were well tolerated. Only one subject developed a ≥4-fold serum hemagglutination-inhibition (HAI) antibody response and a final titer of ≥1∶40 four weeks after dose two and only five subjects developed a neutralizing antibody rise and a final titer of ≥1∶40 in tests performed at a central laboratory. Four of the five were given the 45 or 90 µg HA dosage. A more sensitive HAI assay at the study site revealed a dose-response with increasing HA dosage but only 36% in the 90 µg HA group developed a ≥4-fold rise in antibody in this test and only one of these achieved a titer of ≥1∶32. Conclusion This inactivated subunit influenza A (H7N7) vaccine was safe but poorly immunogenic in humans. Trials Registration ClinicalTrials.gov NCT00546585

Antibody Correlates and Predictors of Immunity to Naturally Occurring Influenza in Humans and the Importance of Antibody to the Neuraminidase

BACKGROUND Serum antibody to the hemagglutinin (HA) of influenza viruses is a correlate and predictor of immunity to influenza in humans; the relative values of other correlates are uncertain. METHODS Serum and nasal secretions (NS) were collected in fall and spring of 2009-2011 from healthy adults who were monitored for acute respiratory illness (ARI). Serum samples were tested for hemagglutination-inhibition (HAI) antibody increase and secretions for virus if ill; enrollment sera were also tested for neuraminidase-inhibiting (NI) antibody and NS for neutralizing (neut), NI, immunoglobulin A (IgA), and immunoglobulin G (IgG) anti-HA antibody. RESULTS Serum anti-HA and anti-neuraminidase (NA) antibody titers to 2009(H1N1) pandemic influenza virus (pH1N1) correlated with titers in NS (including IgA and IgG antibody). Increasing anti-HA and anti-NA titers in serum and NS tests all correlated with reducing infection and infection-associated illness. Multivariate analyses indicated serum HAI and NI each independently predicted immunity to infection and infection-associated illness. Only serum NI independently predicted reduced illness among infected subjects. CONCLUSIONS Increasing anti-HA and NA antibody in serum and secretions correlated with reducing pH1N1 influenza virus infection and illness in healthy young adults. Both anti-HA and anti-NA antibody are independent predictors of immunity to influenza; ensuring induction of both by vaccination is desirable.

A High Dosage Influenza Vaccine Induced Significantly More Neuraminidase Antibody than Standard Vaccine among Elderly Subjects

Antibody to the neuraminidase (NA) antigen of influenza viruses has been shown to correlate with immunity to influenza in humans and animal models. In a previous report, we showed that an inactivated influenza vaccine containing 60microg of the hemagglutinin (HA) of each strain induced significantly more serum anti-HA antibody among elderly persons than did the standard vaccine containing 15microg of the HA of each component. We developed a lectin-based assay for anti-NA antibody and used it to measure anti-NA antibody responses among subjects who had participated in that study. The high dosage vaccine contained eight times as much NA activity as the standard vaccine and induced a significantly higher frequency of antibody responses and higher mean postvaccination anti-NA titers to the N1 and N2 of the A/H1N1 and A/H3N2 viruses in the vaccines than did the standard vaccine. Ensuring an increased antibody response to the NA antigen in inactivated influenza virus vaccines should increase the protection against influenza. An increased quantity of the NA antigen in the vaccine will ensure an increased response.

Integrative genomic analysis of the human immune response to influenza vaccination

Identification of the host genetic factors that contribute to variation in vaccine responsiveness may uncover important mechanisms affecting vaccine efficacy. We carried out an integrative, longitudinal study combining genetic, transcriptional, and immunologic data in humans given seasonal influenza vaccine. We identified 20 genes exhibiting a transcriptional response to vaccination, significant genotype effects on gene expression, and correlation between the transcriptional and antibody responses. The results show that variation at the level of genes involved in membrane trafficking and antigen processing significantly influences the human response to influenza vaccination. More broadly, we demonstrate that an integrative study design is an efficient alternative to existing methods for the identification of genes involved in complex traits. DOI: http://dx.doi.org/10.7554/eLife.00299.001

Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses. Design To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM). Results Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures. Summary Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.

Randomized Comparative Study of the Serum Antihemagglutinin and Antineuraminidase Antibody Responses to Six Licensed Trivalent Influenza Vaccines

BACKGROUND Serum antibody to the hemagglutinin (HA) surface protein of influenza virus induced by influenza vaccination is a correlate of protection against influenza. The neuraminidase (NA) protein is also on the surface of the virus; antibody to it has been shown to impair virus release from infected cells and to reduce the intensity of influenza infections in animal models and in humans challenged with infectious virus. Recently we have shown that NA inhibiting antibody can independently contribute to immunity to naturally-occurring influenza immunity in the presence of antibody to the HA. PURPOSE The present study was conducted to evaluate induction of antibody to the NA and the HA by commercially available influenza vaccines. METHODS Healthy young adults were vaccinated with one of five commercially available trivalent inactivated vaccines or live influenza vaccine. Frequencies of serum antibody and fold geometric mean titer (GMT) increases four weeks later were measured to each of the three vaccine viruses (A/H1N1, A/H3N2, B) in hemagglutination-inhibition (HAI) and neutralization (neut) assays. Frequency and fold GMT increase in neuraminidase-inhibition (NI) antibody titers were measured to the influenza A viruses (A/H1N1, A/H3N2). RESULTS No significant reactogenicity occurred among the vaccinated subjects. The Fluvirin inactivated vaccine induced more anti-HA antibody responses and a higher fold GMT increase than the other inactivated vaccines but there were no major differences in response frequencies or fold GMT increase among the inactivated vaccines. Both the frequency of antibody increase and fold GMT increase were significantly lower for live vaccine than for any inactivated vaccine in HAI and neut assays for all three vaccine viruses. Afluria inactivated vaccine induced more N1 antibody and Fluarix induced more N2 antibody than the other vaccines but all inactivated vaccines induced serum NI antibody. The live vaccine failed to elicit any NI responses for the N2 NA of A/H3N2 virus and frequencies were low for the N1 of A/H1N1 virus. CONCLUSIONS Trivalent inactivated influenza vaccines with similar HA dosage induce similar serum anti-HA antibody responses in healthy adults. Current inactivated vaccines all induce serum anti-NA antibody to the N1 and N2 NA proteins but some are better than others for N1 or N2. The live vaccine, Flumist, was a poor inducer of either anti-HA or anti-NA serum antibody compared to inactivated vaccine in the healthy adults. In view of the capacity for contributing to immunity to influenza in humans, developing guidelines for NA content and induction of NA antibody is desirable.

Host Transcriptional Response to Influenza and Other Acute Respiratory Viral Infections – A Prospective Cohort Study

To better understand the systemic response to naturally acquired acute respiratory viral infections, we prospectively enrolled 1610 healthy adults in 2009 and 2010. Of these, 142 subjects were followed for detailed evaluation of acute viral respiratory illness. We examined peripheral blood gene expression at 7 timepoints: enrollment, 5 illness visits and the end of each year of the study. 133 completed all study visits and yielded technically adequate peripheral blood microarray gene expression data. Seventy-three (55%) had an influenza virus infection, 64 influenza A and 9 influenza B. The remaining subjects had a rhinovirus infection (N = 32), other viral infections (N = 4), or no viral agent identified (N = 24). The results, which were replicated between two seasons, showed a dramatic upregulation of interferon pathway and innate immunity genes. This persisted for 2-4 days. The data show a recovery phase at days 4 and 6 with differentially expressed transcripts implicated in cell proliferation and repair. By day 21 the gene expression pattern was indistinguishable from baseline (enrollment). Influenza virus infection induced a higher magnitude and longer duration of the shared expression signature of illness compared to the other viral infections. Using lineage and activation state-specific transcripts to produce cell composition scores, patterns of B and T lymphocyte depressions accompanied by a major activation of NK cells were detected in the acute phase of illness. The data also demonstrate multiple dynamic gene modules that are reorganized and strengthened following infection. Finally, we examined pre- and post-infection anti-influenza antibody titers defining novel gene expression correlates.

Prior Infections With Seasonal Influenza A/H1N1 Virus Reduced the Illness Severity and Epidemic Intensity of Pandemic H1N1 Influenza in Healthy Adults

Preexisting antibody, responses to seasonal and pandemic 2009 vaccine, a low infection-to-illness ratio, and shortened illnesses indicated preexisting immunity to pandemic H1N1 virus that caused the 2009 epidemic to be mild in healthy adults.

Contrasting effects of type I interferon as a mucosal adjuvant for influenza vaccine in mice and humans.

To identify an adjuvant that enhances antibody responses in respiratory secretions to inactivated influenza virus vaccine (IVV), a comparison was made of responses to intranasal vaccinations of mice with IVV containing monophosphoryl lipid A (MPL), type I interferon (IFN) or cholera toxin B (CTB). Antibody in nasal secretions and lung wash fluids from mice was increased after vaccination and lung virus was significantly reduced after challenge to a similar level in each adjuvant group. Interferon was selected for a trial in humans. Trivalent inactivated influenza vaccine was given intranasally to healthy adult volunteers alone or with 1 million units (Mu) or 10 Mu of alpha interferon. Vaccinations were well tolerated but neither serum hemagglutination-inhibiting nor neutralizing antibody responses among the vaccine groups were significantly different. Similarly, neither neutralizing nor IgA antibody responses in nasal secretions were significantly different. Thus, despite exhibiting a significant adjuvant effect in mice, interferon did not exhibit an adjuvant effect for induction of antibody in respiratory secretions of humans to inactivated influenza virus vaccine given intranasally.